This invention relates to an assay or detection method which employs an enzyme linked chemiluminescent endpoint signalling system for the detection and measurement of compounds of interest in assay systems employing ligand binding techniques.
Such ligand binding techniques depend upon the facility inherent in biological molecules such as receptors, antibodies and nucleic acids to bind with a high degree of specificity their respective analogous partner ligand. Owing to this specificity, such techniques have found widespread application in the detection and measurement of many entities ranging from simple chemicals to complex biological molecules, including peptides, proteins, carbohydrates and nucleic acids. Consequently the technique of ligand binding has become one of the most important tools for investigation and assay, and has thus found universal application.
In such ligand binding systems, the specific binding reaction occurs when the ligand is presented to the ligand partner compound. Examples include the antibody-antigen reaction, and the hybridisation of complementary nucleic acid sequences. A key feature inherent to all ligand binding assay systems is that, in order to monitor the progress of such binding and thus to obtain a qualitative and/or quantitative indication of the degree of such binding, it is necessary to label, either directly or indirectly, at least one of the ligand partners participating in the ligand binding reaction. This labelled ligand can then be employed to generate a measurable signal by which the reaction is monitored. The relative quantity of signal generated by the labelled ligand will be proportional to the quantity of labelled ligand present and thus can serve to indicate the concentration of the labelled ligand. Examples of such signal generators include radioactive nuclides (.sup.125 I, .sup.3 H, .sup.14 C, .sup.32 P), chemiluminescent or fluorescent compounds (acridinium esters, lanthanide chelates) and enzymes (peroxidase, phosphatase).
Such labels are synthesised by chemically coupling the signal generator `label` to the ligand so that they do not perturb its binding characteristics but enable its presence to be measured by the appropriate detection technology. The choice of signal generating labels is influenced by factors such as ease and sensitivity of detection, and the ability to incorporate readily such compounds chemically into the particular ligand. Classically the first class of compounds to be so employed were radionuclides. These can be incorporated into biological molecules and render themselves detectable by virtue of their radioactive emissions which can be monitored by appropriate technology such as scintillation counting and high energy photon sensitive chemical films. However, radioactive moieties suffer from problems associated with their radioactive decay such as safety and instability, as well as limited sensitivity of detection with certain nuclides. Consequently alternative non-isotopic signal generating systems have been sought. These non-isotopic labels are chemically linked to one of the components participating in the ligand binding reaction.
Examples of such alternative non-isotopic signal generating systems include fluorescence, where the output signal is generated by the excitation of a suitable molecular adduct, chemiluminescence where the output signal is generated by chemical modification of the signal compound, or enzymes where there occurs an enzyme dependent secondary generation of signal such as the formation of a coloured product from a non-coloured substrate.
British Patent Specification 2 112 779 describes techniques where acridinium salts have been successfully employed as direct signal generating labels in ligand binding assays. Here the acridinium salt is covalently coupled directly to the ligand. Following the assay procedure the luminescence is measured by subjecting the acridinium salt, linked to the ligand specifically captured in the ligand binding complex, to exposure to peroxide in strong alkali (pH 14). Under these conditions an acridinium ester bond, for example, is broken leading to the formation of an excited product molecule which relaxes to its ground state with loss of energy in the form of photons. These photons can then be quantified by standard luminometric techniques. Typically, though not exclusively, the release of photons is rapid with the signal generating reaction being completed within 1-2 seconds when excess quantities of initiating alkaline peroxide are present. In order for this reaction to be monitored, the addition of one or more initiator compounds must necessarily take place when the reactants are in physical proximity to the luminometric device. This can present limitations to its application, particularly when detecting the signal from labelled ligand captured on a flat surface such as a gel, membrane or tissues. In consequence, despite its very high sensitivity, the employment of acridinium salt luminescence has until now been restricted to ligand binding systems where reactants are enclosed in tubes which can be placed in luminometers prior to in situ addition of initiator reagents.
In the case of enzymes as primary signal generators, the action of the enzyme on an appropriate substrate may itself lead to the generation of a secondary signal which is, for example, chemiluminescent or fluorescent in nature. In this situation the role of the labelling enzyme is either direct, that is to convert the substrate itself from an inactive to an active and therefore detectable form, or indirect, that is to convert the substrate from an inactive to active substance which is itself an initiator or co-factor in the conversion of an inactive to active compound. An example of the former is the direct action of alkaline phosphatase on stable dioxetanes, where removal of a phosphate group renders the dioxetanes unstable with a consequent release of quantifiable luminescence. An example of the latter is the indirect action of peroxidase on luminol where luminescence is generated from enzyme catalysed production of active oxygen species by breakdown of peroxide substrate. In both these examples, after a suitable incubation period, luminescence is relatively long lived as it depends upon the generation of product by the signal enzyme, a process which is essentially continuous until the available substrate is consumed. Of those systems which have found the most widespread application, the signal intensity is inherently low thus limiting the scope of application; in order to overcome this problem, such systems require the use of enhancers (for example, para-iodo-phenol) or amplifiers (for example, fluorescent polymers) thereby increasing the complexity of these signal generating methods.
Ligand binding assays employing oxidase enzymes such as glucose oxidase as signal generating labels have been described previously. In these systems the presence of the ligand-linked oxidase enzyme is monitored indirectly by coupling the reaction to peroxidase enzymes such as horseradish peroxidase to generate active oxygen species which combine with a suitable colourless soluble reducing agent to produce a coloured product. This technique however suffers from poor detectability.
Arakawa et al (Clinical Chemistry vol 31, pp 430-434, 1984) describe a ligand binding assay using glucose oxidase coupled bis-oxalate ester generated luminescence employing a fluorescent dye. Alternatively a method has been described by Kiriyama et al (Clinica Chimica Acta vol 220, pp 201-209, 1993) where the peroxide produced from glucose oxidase linked ligand is monitored by its capacity to generate luminol chemiluminescence in the presence of ferricyanide.
However both these methods are insensitive and extensive duration (overnight) of pre-incubation of the enzyme linked ligand with glucose substrate is required to generate sufficiently detectable concentrations of peroxide.
The inventors have developed a method which benefits from the high photon emission of chemiluminescent acridinium compounds and their analogs, without requiring the lengthy pre-incubation periods of typical enzyme linked assays, and whilst providing a sustained photon emission as compared with the more rapid light emission normally associated with chemiluminescent acridinium compounds and their analogues. The sustained emission means that the high sensitivity of chemiluminescent acridinium compounds and their analogues may now be used for gels, membranes and tissues, where many sites may be assayed simultaneously and the photon emission recorded photographically or by other imaging methods. Further, the sustained emission does not require that the reagents are applied to all the gel sites simultaneously. The shortened incubation period means that assays may be carried out in real time, rather than overnight.
The methods may however be used in many applications other than on gels, membranes or tissues.